Quantitative Analysis of Cell and Tissue Shape During Mouse Cranial Neural Tube Closure

Materials and reagents

Biological materials

1. Mouse embryos at timepoints between embryonic day 7.5 (E7.5) and E8.75 (0–9 somites) with resected yolk and amniotic sacs fixed in 4% paraformaldehyde (see section A for recommended fixation conditions and embryo staging)

Reagents

1. 10× phosphate-buffered saline (PBS) (Fisher, catalog number: BP399-500); an equivalent solution can be prepared from salts, as in [26]

2. Triton X-100 (ThermoFisher, catalog number: A16046.AP)

3. 4% paraformaldehyde in PBS (e.g., Fisher, catalog number: AAJ61899AP), aliquoted in 1 mL tubes and stored at -20 °C until use or up to 1 year

4. Bovine serum albumin (BSA) (e.g., Fisher, catalog number: BP9703100)

5. Agarose, low melt (ThermoFisher, catalog number: H26417.14)

6. Rabbit anti-N-cadherin (Cell Signaling Technology, catalog number: 13116S)

7. Fluorescent conjugated anti-mouse secondary antibodies (e.g., Donkey anti-mouse Alexa488) (Invitrogen, catalog number: A32787)

8. Fluorescent conjugated phalloidin (e.g., Alexa546 conjugated phalloidin) (ThermoFisher, catalog number: A22283)

Solutions

1. 1× PBS + 0.1% Triton X-100 (PBTriton) (see Recipes)

2. Blocking solution (see Recipes)

Recipes

1. PBTriton

This solution is stable for up to one year at 4 °C.

2. Blocking solution

As an alternative, you can buy premade 10% BSA in PBS blocking solution (e.g., ThermoFisher, catalog number: 37525) and add 0.1% Triton X-100. This is stable and—with the addition of detergent—exhibits no biological contamination in our experience for up to one year at 4 °C. If biological contamination is a concern, a small amount of preservative (e.g., 0.05% sodium azide) can be added without impacting downstream use.

Make solutions in 1,000 mL glass bottles. Triton should be added after diluting the 10× PBS stock with water. At stock concentration, it is quite viscous, so cutting a small portion off a pipette tip can help in transferring. Use a magnetic stir bar and plate to thoroughly incorporate Triton into the solution (~30 min).

Laboratory supplies

1. 35 mm untreated plastic or glass Petri dishes (e.g., Fisher, catalog number: FB0875711YZ, though this can be any small Petri dish)

2. Plastic transfer pipettes (e.g., Fisher, catalog number: 13-711-7M)

Note: Ensure that the mouth of the pipette is large enough in diameter to allow the embryo to enter the pipette; if the diameter is too narrow, a pair of scissors can be used to cut off the tip.

3. 24-well untreated plastic well plates (e.g., Genesee Scientific, catalog number: 25-103) or 2 mL Epi tubes (e.g., Genesee Scientific, catalog number: 24-283) for fixing and staining embryos

4. 25 mm circular #1 or #1.5 cover glasses for imaging dishes (e.g., Fisher, catalog number: 50-948-978)

5. Square or rectangular cover glass cut or broken into <10 mm squares

6. Molykote vacuum grease (Fisher, catalog number: 14-635-5D)

7. 1 mL Luer Lock syringe (e.g., Fisher, catalog number: 14-823-30) with a 200 μL pipette tip screwed onto the Luer Lock

Equipment

1. Watchmakers forceps, ideally #55 or #5 (e.g., Fine Science Tools, catalog number: 11295-51)

2. Orbital shaker or nutator

3. Attofluor reusable aluminum imaging dishes (ThermoFisher, catalog number: A7816)

4. Dissecting stereomicroscope or macroscope equipped with brightfield camera (e.g., Zeiss Lumar, model: V12)

5. Confocal laser scanning microscope in inverted light path configuration and equipped with lasers appropriate to the fluorescent conjugates of your antibodies (e.g., Zeiss, model: LSM 980)

6. Computer for image analysis

Software and datasets

1. FIJI/ImageJ (https://fiji.sc/; NIH, and worldwide contributors, see [27,28], free to use)

2. The MorphoLibJ plugin for FIJI/ImageJ (https://github.com/ijpb/MorphoLibJ, INRA-IJBP modeling lab, see [29], free to use)

3. Cellpose (https://www.cellpose.org/, see [30], free to use); alternatively, cell-wise segmentation program (e.g., SeedWater segmenter, David Mashburn/Hutson Lab, see [31], free to use)

Note: We routinely update our FIJI/ImageJ distributions, the MorphoLibJ plugin, and the Cellpose software; to date, it has not impacted the workflow presented below. As of this writing, the versions we used were: FIJI/ImageJ- ImageJ 2 16.0/1.54p (running on Java 1.8.0_172); MorphoLibJ-1.6.5; Cellpose-4.1. For Cellpose, we recommend starting with the pretrained CP (cyto fluorescent) model and retraining it on manually corrected data from 10 embryos for any given stain to increase the accuracy of the automatic segmentation.

Procedure